| Summary: | 碩士 === 淡江大學 === 生命科學研究所碩士班 === 95 === Capsulin is a basic helix-loop-helix transcription factor involved in the regulation of cell differentiation. In chicken and mice, capsulin is expressed in mesenchymal cells and developing branchiomeric muscles derived from the non-segmented head mesoderm. However, the biological functions of capsulin during craniofacial myogenesis was little known. Here, we used zebrafish as a model to study the biological functions of capsulin during early development because of their well-defined developmental stages and genetic background. Using reverse transcription-polymerase chain reaction (RT-PCR), capsulin cDNA was cloned from the mRNA of zebrafish embryos. The deduced zebrafish Capsulin amino acid sequence revealed a 176-amino acid polypeptide containing a helix-loop-helix motif. After sequences comparison, we found that the zebrafish capsulin polypeptide shares sequence identities of 75, 75, 76, 73 and 71% with the reported capsulin of human, bovine, mouse, chicken and Xenopus, respectively. Whole-mount in situ hybridization experiments using capsulin anti-sense riboprobe revealed that capsulin was first detected at the central parts of the branchial arches of the 24 hours postfertilization (hpf) embryos. At later stages, capsulin expression was detected in the heart , pectoral fin buds and both terminal ends of each branchial arch. Double whole-mount in situ hybridization revealed that capsulin expression domains were mainly overlapped with those of dlx2 and hand2 (neural crest cells’ markers), indicating that capsulin should be a novel neural crest cells marker. To further investigate the biological functions of capsulin during early embryogenesis, we used morpholino to knock down endogenous capsulin translation. Subtle changes of cartilages and muscle fibers were easily observed when embryos were stained with Alcian blue and monoclonal antibody F59, respectively. Our data showed that knock down of capsulin led to loss of all cranial muscles. In addition, cartilages abnormalities were observed in capsulin-morphants, including loss of ceratobrachial, and enlarged angles of ceratohyal. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were also performed, and revealed that large amounts of cell apoptosis appeared in the head region of capsulin-morphants. Furthermore, whole-mount in situ hybridization experiments were carried out using anti-sense dlx2, sox9a (cranial neural crest cells’ marker) and myf5 (myocyte-specific marker) as riboprobes. Results showed that capsulin-morphants displayed reduction of cranial neural crest cells which are required for 2nd and 3rd arches formation; myf5 transcripts were down-regulated at the precursor cells of craniofacial-, pectoral fin- and hypaxial-muscles.In addition，we found that co-injection the capsulin-MO with the mRNAs of myf5、myoD、myf5 and myoD led to recovery of no cranialmuscles phenotype.These observations clearly indicated that knock down of capsulin translation induced parts of cranial neural crest cells apoptosis and consequently affected cartilages and craniofacial muscles formation. From these result, we concluded that capsulin is required for craniofacial organization, especially for cranial myogenesis in zebrafish embryos.
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